Impaired immune functions leading to primary immunodeficiencies often correlate with paradoxical autoimmune complications. Patients with primary immunodeficiencies provide rare opportunities to study the impact of specific defective genes on the regulation of B cell tolerance and the removal of developing autoreactive B cells in humans. Alterations in B cell receptor (BCR) signaling pathways in patients lacking functional BTK, CD19, or molecules mediating TLR signaling such as IRAK4, MyD88, and UNC93B result in a defective central checkpoint and a failure to counterselect developing autoreactive B cells (1, 2). Indeed, the binding of self-antigens to autoreactive BCRs and TLRs fail to induce tolerance mechanisms due to increase receptor signaling thresholds in all these patients' B cells and autoreactive B cells leaks from the bone marrow into the periphery (1, 2). Our latest investigations revealed that central B cell tolerance defects are primary to many autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and type 1 diabetes (T1D) and result from genetic factors segregating with autoimmunity and which encode variants interfering with BCR signaling (3-5). However, we recently found that patients with multiple sclerosis (MS) only displayed peripheral B cell tolerance defects whereas central B cell tolerance was often established normally. Similar observations were observed in CD40L- and MHC class II-deficient patients, who pointed to a potential role for regulatory T (Treg) cells and serum B-cell activating factor (BAFF) in the removal of developing autoreactive B cells in the periphery (6). Interestingly, Treg cell functions have been reported to be defective in MS patients. Hence, Treg cells as second axis controlling the establishment and the maintenance of B cell tolerance in the periphery is proposed to be analyzed in this R21 application by studying immune deficiency, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients, who display a defective Treg cell compartment and suffer from autoimmune condition and the secretion of many self-reactive antibodies. The long range goal of the proposed research is to determine the mechanisms that regulate peripheral B cell tolerance in healthy humans but may be defective in IPEX and APECED patients. The working hypothesis is that genetic defects impacting Treg cell functions not only interfere with the removal of autoreactive B cells in the periphery but may lead to their activation, thereby potentially favoring the development of autoimmunity. Similarly to CD40L-deficient patients, we expect to find that FOXP3- and AIRE-deficient patients with abnormal Treg cell functions will display normal central yet defective peripheral B cell tolerance checkpoints. We will also assess the mechanisms by which Treg cells may induce autoreactive B cell death and contribute to B cell homeostasis regulation. Altogether, the proposed study intends to demonstrate a role for Treg cells in the restraining autoreactive B cells in the periphery.